Physiological Changes And Proteomics Of Wide Species Of Chrysanthemum In Response To Drought Stress

Chrysanthemum(Chrysanthemum morifolium), as one of the four most popular cut flower over the world and one of the ten most famous flowers in China, provides high ornamental and economic value, which possess the important status on flower industry. Water scarcity caused by drought stress has gradually become one of the main factors limiting the chrysanthemum production and landscaping applications. Studying the mechanisms for drought tolerance and the adaptation and regulatory mechanisms to drought in chrysanthemum is crucial for selecting and breeding drought-tolerant cultivars, which could be an important strategy to cope with the water shortage and the deterioration of the ecological environment. Wild species of Chrysanthemum often carry important resistance genes and are important resource for resistant breeding materials. Tolerant (C. japonense) and sensitive (C. nankingense) wild species were compared to investigate mechanisms of drought tolerance. We compared the leaf epidermal surface morphology of the two species. We also compared their physiological and metabolic changes in response to drought stress induced by polyethylene glycol. A proteomic approach was used to identify drought-responsive proteins in leaves of C. japonense. The main results are as follows:1. The response of C. japonense and C. nankingense to drought stress induced by polyethylene glycol was characterized by leaf water status, leaf surface morphology and cuticular wax (quantity and composition), the activity of antioxidant enzymes, the extent of membrane lipid peroxidation, the accumulation of proline, photosynthesis performance and abscisic acid (ABA) accumulation. We found that C. japonense had denser trichomes and more cuticular wax on the leaf epidermis than C. nankingense. Although the stress induced wilting in both species, symptoms were more appeared earlier in the intolerant C. nankingense and all its leaves shrunk to wither. The more tolerant species C. japonense maintained its water status more effectively than C. nankingense, probably because its leaves form more cuticular wax and are able to accumulate higher levels of ABA. Membrane damage, as measured by electrolyte leakage and malondialdehyde accumulation, was less severe in C. japonense, which was also able to generate higher levels of free proline after a ten hour exposure to stress. Superoxide dismutase activity was higher in C. japonense than in C. nankingense, as was that of catalase and ascorbate peroxidase during the later part of the stress episode, but levels of peroxidase were not differentiated at the end of the stress period. Thus the superior response of C. japonense also reflects a more adapted system of osmoprotection and antioxidation. Photosynthetic parameters in both species, such as net photosynthetic rate, stomatal conductance, transpiration rate, intercellular CO2concentratio, maximum efficiency of PSII photochemistry and chlorophyll content, were decreased in both species, but the values of C. japonense were significant grater than C. nankingense at the end of the stress period. As a result, photosynthesis was compromised less by drought stress in C. japonense than in C. nankingense. We found that the superior tolerance of C. japonense likely flows from a combination of its better developed trichome layer, higher cuticular wax content, more rapid and abundant accumulation of ABA, more flexible photosynthesis capacity, and more effective osmoprotective and antioxidative system compared to the intolerant species.2. In order to improve our understanding of the complex mechanisms involved in the response of chrysanthemum to drought stress, a proteomic approach was used to identify drought-responsive proteins in leaves of a drought-tolerant chrysanthemum (C. japonense). Two dimensional gels electrophoresis (2-DE) was used to compare differences in protein abundance between control and stressed plants. Thirty-seven differently expressed spots were selected to be excised and analyzed using MALDI-TOF/TOF-MS, and finally twenty-seven protein spots were confidently identified according to NCBInr database. The majority of identified proteins were classified into functional categories that include photosynthesis (27%), energy metabolism (15%), protein synthesis (11%), stress response and defense (8%), nucleic acid metabolism (8%) and signal transduction (4%). We successfully cloned nine genes encoding these putative differentially accumulated proteins and analyzed their mRNA levels, seven of which were in accordance with the results of2-DE. This is the first report of proteomic study drought-responsive protein in chrysanthemum and we found some differently expressed proteins, like chloroplast manganese stabilizing protein, phenylalanine ammonia-lyase, glutathione S-transferase, Rubisco activase, retrotransposon protein, etc. We found that C. japonense responded to drought stress by triggering the expression some stress-induced genes; improving the activity of antioxidant enzymes to reduce ROS; maintaining the activity of photosynthetic enzymes to keep photosynthesis; strengthening energy metabolic pathways, to maintain the supply of energy and substances in cells; maintaining the essential physiological metabolism to survive under drought.